Reducing Immune Inhibition Induced by SIGLEC-15

ABSTRACT

The present disclosure relates to a modified cell comprising a polynucleotide encoding a secretable scFv binding SIGLEC-15 and/or encoding a dominant negative form of CD44. In embodiments, the modified cell further comprises an antigen-binding molecule, which for example, is a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/837,932, filed Apr. 24, 2019, U.S. Provisional Application62/837,920, filed Apr. 24, 2019, and U.S. Provisional Application62/845,631, filed May 9, 2019, which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING INFORMATION

A computer readable textfile, entitled “Sequence Listing.txt,” createdon or about Apr. 22, 2020, with a file size of about 312 KB, containsthe sequence listing for this application and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions of agent-sensing immunecells and uses thereof in the treatment of diseases, including cancer.

BACKGROUND

T cell therapies have demonstrated efficacy and curative potential fortreating cancers. For example, Chimeric Antigen Receptor (CAR) T celltherapy has achieved good clinical efficacy in cancer, such asB-ALL/CLL/lymphoma. However, use of these therapies has been limited bythe presence of an immunosuppressive microenvironment. Theimmunosuppressive microenvironment includes immune inhibition on, forexample, T cells, which is induced by immune inhibitors such as PD1.PD-1 is a negative coregulatory receptor on T cells andantigen-presenting cells. The PD-L1 is expressed by several cell types(e.g., tumor cells and other tissue cells), and appears to bedynamically regulated by the immune microenvironment. Therefore, thereis a need to address immune tolerance induced by these immune inhibitorsto improve the efficacy of T cell therapies.

SUMMARY

Embodiments relate to a modified cell comprising a polynucleotideencoding a secretable scFv binding SIGLEC-15 and/or encoding a dominantnegative form of CD44. In embodiments, the modified cell comprises anantigen-binding molecule, which, for example, is a CAR comprising anantigen-binding domain, a transmembrane domain, and an intracellularsignaling domain. Embodiments relate to a fusion protein comprising anscFv binding SIGLEC-15, a linker, an extracellular domain, atransmembrane domain, and a cytoplasmic domain, wherein thetransmembrane domain is selected from a group consisting of atransmembrane domain of a receptor of IL15, IL2, IL7, IL6, IL12, IL18,IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the cytoplasmicdomain is selected from a group consisting of a cytoplasmic domain ofreceptor of the receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23,IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the extracellular domain isselected from a group consisting of an extracellular domain of thereceptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα,TNFβ, IFNα, and IFNβ. Embodiments also relate to a fusion proteincomprising an extracellular domain, a transmembrane domain, and acytoplasmic domain, the transmembrane domain being or comprising thetransmembrane domain of Notch.

This Summary is not intended to identify key features or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is described with reference to the accompanyingfigures. The use of the same reference numbers in different figuresindicates similar or identical items.

FIG. 1 shows a schematic diagram of an exemplary fusion protein.

FIG. 2 shows a schematic diagram of another exemplary fusion protein.

FIG. 3 shows a schematic diagram of an exemplary CAR molecule and afusion protein.

FIG. 4 shows a schematic diagram of an exemplary CAR molecule and adominant negative CD44.

FIG. 5 shows schematic diagrams of exemplary constructs of dominantnegative CD44.

FIG. 6 shows flow cytometry results of CART cells that have beencultured to Day 5.

FIGS. 7, 8, 9, and 10 show CAR expression and functions of T cellscomprising a CAR and T cells comprising a CAR and secretable SIGLEC-15scFv.

FIG. 11 shows a scheme of administering an agent (e.g., an inhibitor orinducer) in exemplary adoptive cell therapy. Arrows indicate the timewhen an agent (e.g., inhibitor or inducer) is administered to a subject,for example, at the peak of cytokine release.

FIG. 12 shows a schematic diagram of exemplary structures of membraneproteins of a modified cell and uses thereof.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any method andmaterial similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “activation,” as used herein, refers to the state of a cellthat has been sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody” is used in the broadest sense and refers tomonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity or function. The antibodies in the presentdisclosure may exist in a variety of forms including, for example,polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab′, and F(ab′)₂fragments; as well as single chain antibodies and humanized antibodies(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies:A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426).

The term “antibody fragments” refers to a portion of a full-lengthantibody, for example, the antigen-binding or variable region of theantibody. Other examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multi-specific antibodies formed from antibodyfragments.

The term “Fv” refers to the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragment consistsof a dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanates six hypervariable loops (3 loops each from the H and L chain)that contribute amino acid residues for antigen-binding and conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv including only three complementaritydetermining regions (CDRs) specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site (the dimer).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. An “antibody light chain,” asused herein, refers to the smaller of the two types of polypeptidechains present in all antibody molecules in their naturally occurringconformations. K and A light chains refer to the two major antibodylight chain isotypes.

The term “synthetic antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage. The term also includes an antibody whichhas been generated by the synthesis of a DNA molecule encoding theantibody and the expression of the DNA molecule to obtain the antibodyor to obtain an amino acid encoding the antibody. The synthetic DNA isobtained using technology that is available and well known in the art.

The term “antigen” refers to a molecule that provokes an immuneresponse, which may involve either antibody production, or theactivation of specific immunologically-competent cells, or both.Antigens include any macromolecule, including all proteins or peptides,or molecules derived from recombinant or genomic DNA. For example, DNAincluding a nucleotide sequence or a partial nucleotide sequenceencoding a protein or peptide that elicits an immune response, andtherefore, encodes an “antigen” as the term is used herein. An antigenneed not be encoded solely by a full-length nucleotide sequence of agene. An antigen can be generated, synthesized or derived from abiological sample including a tissue sample, a tumor sample, a cell, ora biological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect associated with a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, decreasein tumor cell proliferation, decrease in tumor cell survival, anincrease in life expectancy of a subject having tumor cells, oramelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells, and antibodies inthe prevention of the occurrence of tumors in the first place.

The term “auto-antigen” refers to an endogenous antigen mistakenlyrecognized by the immune system as being foreign. Auto-antigens includecellular proteins, phosphoproteins, cellular surface proteins, cellularlipids, nucleic acids, glycoproteins, including cell surface receptors.

The term “autologous” is used to describe a material derived from asubject that is subsequently re-introduced into the same subject.

The term “allogeneic” is used to describe a graft derived from adifferent subject of the same species. As an example, a donor subjectmay be related or unrelated to the recipient subject, but the donorsubject has immune system markers that are similar to the recipientsubject.

The term “xenogeneic” is used to describe a graft derived from a subjectof a different species. As an example, the donor subject is from adifferent species than a recipient subject, and the donor subject andthe recipient subject can be genetically and immunologicallyincompatible.

The term “cancer” is used to refer to a disease characterized by therapid and uncontrolled growth of aberrant cells. Cancer cells can spreadlocally or through the bloodstream and lymphatic system to other partsof the body. Examples of various cancers include breast cancer, prostatecancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “includes” and “including” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

The phrase “consisting of” is meant to include, and is limited to,whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory and that no other elements may be present.

The phrase “consisting essentially of” is meant to include any elementlisted after the phrase and can include other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules, or theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

The term “corresponds to” or “corresponding to” refers to (a) apolynucleotide having a nucleotide sequence that is substantiallyidentical or complementary to all or a portion of a referencepolynucleotide sequence or encoding an amino acid sequence identical toan amino acid sequence in a peptide or protein, or (b) a peptide orpolypeptide having an amino acid sequence that is substantiallyidentical to a sequence of amino acids in a reference peptide orprotein.

The term “co-stimulatory ligand,” refers to a molecule on anantigen-presenting cell (e.g., an APC, dendritic cell, B cell, and thelike) that specifically binds a cognate co-stimulatory molecule on a Tcell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, includingat least one of proliferation, activation, differentiation, and othercellular responses. A co-stimulatory ligand can include B7-1 (CD80),B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatoryligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40,CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6,ILT3, ILT4, HVEM, a ligand for CD7, an agonist or antibody that bindsthe Toll ligand receptor, and a ligand that specifically binds withB7-H3. A co-stimulatory ligand also includes, inter alia, an agonist oran antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds CD83.

The term “co-stimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a co-stimulatory ligand,thereby mediating a co-stimulatory response by the T cell, such asproliferation. Co-stimulatory molecules include an MHC class I molecule,BTLA, and a Toll-like receptor.

The term “co-stimulatory signal” refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

The terms “disease” and “condition” may be used interchangeably or maybe different in that the particular malady or condition may not have aknown causative agent (so that etiology has not yet been worked out),and it is therefore not yet recognized as a disease but only as anundesirable condition or syndrome, wherein a more or less specific setof symptoms have been identified by clinicians. The term “disease” is astate of health of a subject wherein the subject cannot maintainhomeostasis, and wherein if the disease is not ameliorated then thesubject's health continues to deteriorate. In contrast, a “disorder” ina subject is a state of health in which the animal is able to maintainhomeostasis, but in which the animal's state of health is less favorablethan it would be in the absence of the disorder. Left untreated, adisorder does not necessarily cause a further decrease in the animal'sstate of health.

The term “effective” refers to adequate to accomplish a desired,expected, or intended result. For example, an “effective amount” in thecontext of treatment may be an amount of a compound sufficient toproduce a therapeutic or prophylactic benefit.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as a template for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene encodes a protein if transcription and translation of mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. Both the coding strand, the nucleotide sequence ofwhich is identical to the mRNA sequence (except that a “T” is replacedby a “U”) and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “exogenous” refers to a molecule that does not naturally occurin a wild-type cell or organism but is typically introduced into thecell by molecular biological techniques. Examples of exogenouspolynucleotides include vectors, plasmids, and/or man-made nucleic acidconstructs encoding the desired protein. With regard to polynucleotidesand proteins, the term “endogenous” or “native” refers tonaturally-occurring polynucleotide or amino acid sequences that may befound in a given wild-type cell or organism. Also, a particularpolynucleotide sequence that is isolated from a first organism andtransferred to a second organism by molecular biological techniques istypically considered an “exogenous” polynucleotide or amino acidsequence with respect to the second organism. In specific embodiments,polynucleotide sequences can be “introduced” by molecular biologicaltechniques into a microorganism that already contains such apolynucleotide sequence, for instance, to create one or more additionalcopies of an otherwise naturally-occurring polynucleotide sequence, andthereby facilitate overexpression of the encoded polypeptide.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by its promoter.

The term “expression vector” refers to a vector including a recombinantpolynucleotide including expression control (regulatory) sequencesoperably linked to a nucleotide sequence to be expressed. An expressionvector includes sufficient cis-acting elements for expression; otherelements for expression can be supplied by the host cell or in an invitro expression system. Expression vectors include all those known inthe art, such as cosmids, plasmids (e.g., naked or contained inliposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses,and adeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “homologous” refers to sequence similarity or sequence identitybetween two polypeptides or between two polynucleotides when a positionin both of the two compared sequences is occupied by the same base oramino acid monomer subunit, e.g., if a position in each of two DNAmolecules is occupied by adenine, then the molecules are homologous atthat position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared ×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences ATTGCC and TATGGC share 50% homology. Acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” refers to a class of proteins, whichfunction as antibodies. The five members included in this class ofproteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibodythat is present in body secretions, such as saliva, tears, breast milk,gastrointestinal secretions and mucus secretions of the respiratory andgenitourinary tracts. IgG is the most common circulating antibody. IgMis the main immunoglobulin produced in the primary immune response inmost subjects. It is the most efficient immunoglobulin in agglutination,complement fixation, and other antibody responses, and is important indefense against bacteria and viruses. IgD is the immunoglobulin that hasno known antibody function but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingthe release of mediators from mast cells and basophils upon exposure tothe allergen.

The term “isolated” refers to a material that is substantially oressentially free from components that normally accompany it in itsnative state. The material can be a cell or a macromolecule such as aprotein or nucleic acid. For example, an “isolated polynucleotide,” asused herein, refers to a polynucleotide, which has been purified fromthe sequences which flank it in a naturally-occurring state, e.g., a DNAfragment which has been removed from the sequences that are normallyadjacent to the fragment. Alternatively, an “isolated peptide” or an“isolated polypeptide” and the like, as used herein, refer to in vitroisolation and/or purification of a peptide or polypeptide molecule fromits natural cellular environment, and from association with othercomponents of the cell.

The term “substantially purified” refers to a material that issubstantially free from components that are normally associated with itin its native state. For example, a substantially purified cell refersto a cell that has been separated from other cell types with which it isnormally associated in its naturally occurring or native state. In someinstances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to a cell that has been separated from the cells with which theyare naturally associated in their natural state. In embodiments, thecells are cultured in vitro. In embodiments, the cells are not culturedin vitro.

In the context of the present disclosure, the following abbreviationsfor the commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. Moreover, the use oflentiviruses enables integration of the genetic information into thehost chromosome, resulting in stably transduced genetic information.HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived fromlentiviruses offer the means to achieve significant levels of genetransfer in vivo.

The term “modulating,” refers to mediating a detectable increase ordecrease in the level of a response in a subject compared with the levelof a response in the subject in the absence of a treatment or compound,and/or compared with the level of a response in an otherwise identicalbut untreated subject. The term encompasses perturbing and/or affectinga native signal or response thereby mediating a beneficial therapeuticresponse in a subject, preferably, a human.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence, ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation.

The term “under transcriptional control” refers to a promoter beingoperably linked to and in the correct location and orientation inrelation to a polynucleotide to control (regulate) the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area such as a solid tumor withina specific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients having asolid tumor or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, pheochromocytomas sebaceous gland carcinoma, papillarycarcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma(such as brainstem glioma and mixed gliomas), glioblastoma (also knownas glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma, and brain metastases).

A solid tumor antigen is an antigen expressed on a solid tumor. Inembodiments, solid tumor antigens are also expressed at low levels onhealthy tissue. Examples of solid tumor antigens and their relateddisease tumors are provided in Table 1.

TABLE 1 Solid Tumor antigen Disease tumor PRLR Breast Cancer CLCA1colorectal Cancer MUC12 colorectal Cancer GUCY2C colorectal Cancer GPR35colorectal Cancer CR1L Gastric Cancer MUC 17 Gastric Cancer TMPRSS11Besophageal Cancer MUC21 esophageal Cancer TMPRSS11E esophageal CancerCD207 bladder Cancer SLC30A8 pancreatic Cancer CFC1 pancreatic CancerSLC12A3 Cervical Cancer SSTR1 Cervical tumor GPR27 Ovary tumor FZD10Ovary tumor TSHR Thyroid Tumor SIGLEC-15 Urothelial cancer SLC6A3 Renalcancer KISS1R Renal cancer QRFPR Renal cancer: GPR119 Pancreatic cancerCLDN6 Endometrial cancer/Urothelial cancer UPK2 Urothelial cancer(including bladder cancer) ADAM12 Breast cancer, pancreatic cancer andthe like SLC45A3 Prostate cancer ACPP Prostate cancer MUC21 Esophagealcancer MUC16 Ovarian cancer MS4A12 Colorectal cancer ALPP Endometrialcancer CEA Colorectal carcinoma EphA2 Glioma FAP Mesotelioma GPC3 Lungsquamous cell carcinoma IL13-Rα2 Glioma Mesothelin Metastatic cancerPSMA Prostate cancer ROR1 Breast lung carcinoma VEGFR-II Metastaticcancer GD2 Neuroblastoma FR-α Ovarian carcinoma ErbB2 Carcinomasb EpCAMCarcinomasa EGFRvIII Glioma—Glioblastoma EGFR Glioma—NSCL cancer tMUC 1Cholangiocarcinoma, Pancreatic cancer, Breast Cancer PSCA pancreas,stomach, or prostate cancer

The term “parenteral administration” of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.),intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” and “individual,” and the like are usedinterchangeably herein and refer to any human, or animal, amenable tothe methods described herein. In certain non-limiting embodiments, thepatient, subject, or individual is a human or animal. In embodiments,the term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, and animals, such as dogs, cats, mice, rats, andtransgenic species thereof.

A subject in need of treatment or in need thereof includes a subjecthaving a disease, condition, or disorder that needs to be treated. Asubject in need thereof also includes a subject that needs treatment forprevention of a disease, condition, or disorder.

The term “polynucleotide” or “nucleic acid” refers to mRNA, RNA, cRNA,rRNA, cDNA or DNA. The term typically refers to a polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes all forms of nucleic acids including single anddouble-stranded forms of nucleic acids.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletions,and substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between, e.g., 90%,95%, or 98%) sequence identity with a reference polynucleotide sequencedescribed herein. The terms “polynucleotide variant” and “variant” alsoinclude naturally-occurring allelic variants and orthologs.

The terms “polypeptide,” “polypeptide fragment,” “peptide,” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues of the same. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues are synthetic non-naturally occurring amino acids, such as achemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The term “polypeptide variant” refers to polypeptides that aredistinguished from a reference polypeptide sequence by the addition,deletion, or substitution of at least one amino acid residue. Inembodiments, a polypeptide variant is distinguished from a referencepolypeptide by one or more substitutions, which may be conservative ornon-conservative. In embodiments, the polypeptide variant comprisesconservative substitutions and, in this regard, it is well understood inthe art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide. Polypeptide variants also encompass polypeptides in whichone or more amino acids have been added or deleted or replaced withdifferent amino acid residues.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence. Theterm “expression control (regulatory) sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

The term “bind,” “binds,” or “interacts with” refers to a moleculerecognizing and adhering to a second molecule in a sample or organismbut does not substantially recognize or adhere to other structurallyunrelated molecules in the sample. The term “specifically binds,” asused herein with respect to an antibody, refers to an antibody whichrecognizes a specific antigen, but does not substantially recognize orbind other molecules in a sample. For example, an antibody thatspecifically binds an antigen from one species may also bind thatantigen from one or more species. But, such cross-species reactivitydoes not itself alter the classification of an antibody as specific. Inanother example, an antibody that specifically binds an antigen may alsobind different allelic forms of the antigen. However, such crossreactivity does not itself alter the classification of an antibody asspecific. In some instances, the terms “specific binding” or“specifically binding,” can be used in reference to the interaction ofan antibody, a protein, or a peptide with a second chemical species, tomean that the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the chemicalspecies; for example, an antibody recognizes and binds a specificprotein structure rather than to any protein. If an antibody is specificfor epitope “A,” the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less. A “decreased” or “reduced” or“lesser” amount is typically a “statistically significant” or aphysiologically significant amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as signaltransduction via the TCR/CD3 complex. Stimulation can mediate alteredexpression of certain molecules, such as downregulation of TGF-β, and/orreorganization of cytoskeletal structures.

The term “stimulatory molecule” refers to a molecule on a T cell thatspecifically binds a cognate stimulatory ligand present on an antigenpresenting cell. For example, a functional signaling domain derived froma stimulatory molecule is the zeta chain associated with the T cellreceptor complex. The stimulatory molecule includes a domain responsiblefor signal transduction.

The term “stimulatory ligand” refers to a ligand that when present on anantigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, andthe like.) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a cell, for examplea T cell, thereby mediating a primary response by the T cell, includingactivation, initiation of an immune response, proliferation, and similarprocesses. Stimulatory ligands are well-known in the art and encompass,inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “therapeutic” refers to a treatment and/or prophylaxis. Atherapeutic effect is obtained by suppression, remission, or eradicationof a disease state or alleviating the symptoms of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or another clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent the development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

The term “treat a disease” refers to the reduction of the frequency orseverity of at least one sign or symptom of a disease or disorderexperienced by a subject.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which an exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed, or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “vector” refers to a polynucleotide that comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding linear polynucleotides, polynucleotides associated with ionicor amphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term alsoincludes non-plasmid and non-viral compounds which facilitate thetransfer of nucleic acid into cells, such as, for example, polylysinecompounds, liposomes, and the like. Examples of viral vectors includeadenoviral vectors, adeno-associated virus vectors, retroviral vectors,and others. For example, lentiviruses are complex retroviruses, which,in addition to the common retroviral genes gag, pol, and env, containother genes with regulatory or structural function. Lentiviral vectorsare well known in the art. Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2, and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu, and nef are deleted making the vector biologically safe.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

A “chimeric antigen receptor” (CAR) molecule is a recombinantpolypeptide including at least an extracellular domain, a transmembranedomain and a cytoplasmic domain or intracellular domain. In embodiments,the domains of the CAR are on the same polypeptide chain, for example achimeric fusion protein. In embodiments, the domains are on differentpolypeptide chains, for example the domains are not contiguous.

The extracellular domain of a CAR molecule includes an antigen-bindingdomain. The antigen-binding domain is for expanding and/or maintainingthe modified cells, such as a CAR T cell or for killing a tumor cell,such as a solid tumor. In embodiments, the antigen-binding domain forexpanding and/or maintaining modified cells binds an antigen, forexample, a cell surface molecule or marker, on the surface of a WBC. Inembodiments, the WBC is a granulocyte, monocyte and or lymphocyte. Inembodiments, the WBC is a lymphocyte, for example, a B cell. Inembodiments, the WBC is a B cell. In embodiments, the cell surfacemolecule of a B cell includes CD19, CD22, CD20, BCMA, CD5, CD7, CD2,CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38,CD138, or CD13. In embodiments, the cell surface molecule of the B cellis CD19, CD20, CD22, or BCMA. In embodiments, the cell surface moleculeof the B cell is CD19.

Modified cells (e.g., T-cells and NK cells) may be derived from a stemcell. The stem cells may be adult stem cells, embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, induced pluripotent stem cells,totipotent stem cells or hematopoietic stem cells. A modified cell mayalso be a dendritic cell, a NK-cell, a B-cell or a T-cell selected fromthe group consisting of inflammatory T-lymphocytes, cytotoxicT-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. Inanother embodiment, Modified cells may be derived from the groupconsisting of CD4+T-lymphocytes and CD8+T-lymphocytes. Prior toexpansion and genetic modification of the cells of the invention, asource of cells may be obtained from a subject through a variety ofnon-limiting methods. T cells may be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Inembodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. Inembodiments, modified cells may be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In embodiments, modified cell is part of a mixed populationof cells which present different phenotypic characteristics.

The term “stem cell” refers to any of certain types of cell which havethe capacity for self-renewal and the ability to differentiate intoother kind(s) of cell. For example, a stem cell gives rise either to twodaughter stem cells (as occurs in vitro with embryonic stem cells inculture) or to one stem cell and a cell that undergoes differentiation(as occurs e.g. in hematopoietic stem cells, which give rise to bloodcells). Different categories of stem cell may be distinguished on thebasis of their origin and/or on the extent of their capacity fordifferentiation into other types of cell. For example, stem cell mayinclude embryonic stem (ES) cells (i.e., pluripotent stem cells),somatic stem cells, Induced pluripotent stem cells, and any other typesstem cells.

The pluripotent embryonic stem cells may be found in the inner cell massof a blastocyst and have high innate capacity for differentiation. Forexample, pluripotent embryonic stem cells may have the potential to formany type of cell in the body. When grown in vitro for long periods oftime, ES cells maintain pluripotency: progeny cells retain the potentialfor multilineage differentiation.

Somatic stem cells may include the fetal stem cells (from the fetus) andadult stem cells (found in various tissues, such as bone marrow). Thesecells have been regarded as having a capacity for differentiation lowerthan that of the pluripotent ES cells—with the capacity of fetal stemcells being greater than that of adult stem cells; they apparentlydifferentiate into only a limited range of types of cell and have beendescribed as multipotent. The ‘tissue-specific’ stem cells normally giverise to only one type of cell. For example, embryonic stem cells may bedifferentiated into blood stem cells (e.g., Hematopoietic stem cells(HSCs)), which may be further differentiated into various blood cells(e.g., red blood cells, platelets, white blood cells, etc.).

Induced pluripotent stem cells (i.e., iPS cells or iPSCs) may include atype of pluripotent stem cell artificially derived from anon-pluripotent cell (e.g., an adult somatic cell) by inducing aexpression of specific genes. Induced pluripotent stem cells are similarto natural pluripotent stem cells, such as embryonic stem (ES) cells, inmany aspects, such as the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability. Induced pluripotent cells may be made from adultstomach, liver, skin cells and blood cells.

In embodiments, the antigen-binding domain for killing a tumor, binds anantigen on the surface of a tumor, for example a tumor antigen or tumormarker. Tumor antigens are proteins that are produced by tumor cellsthat elicit an immune response, particularly T cell mediated immuneresponses. Tumor antigens are well known in the art and include, forexample, tumor associated MUC1 (tMUC1), a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, surviving, telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, CD19, andmesothelin. For example, when the tumor antigen is CD19, the CAR thereofcan be referred to as CD19CAR, which is a CAR molecule that includes aantigen-binding domain that binds CD19.

In embodiments, the extracellular antigen-binding domain of a CARincludes at least one scFv or at least a single domain antibody. As anexample, there can be two scFvs on a CAR. The scFv includes a lightchain variable (VL) region and a heavy chain variable (VH) region of atarget antigen-specific monoclonal antibody joined by a flexible linker.Single chain variable region fragments can be made by linking lightand/or heavy chain variable regions by using a short linking peptide(Bird et al., Science 242:423-426, 1988). An example of a linkingpeptide is the GS linker having the amino acid sequence (GGGGS)₃ (SEQ IDNO: 120), which bridges approximately 3.5 nm between the carboxyterminus of one variable region and the amino terminus of the othervariable region. Linkers of other sequences have been designed and used(Bird et al., 1988, supra). In general, linkers can be short, flexiblepolypeptides and preferably comprised of about 20 or fewer amino acidresidues. The single chain variants can be produced either recombinantlyor synthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

The cytoplasmic domain of the CAR molecules described herein includesone or more co-stimulatory domains and one or more signaling domains.The co-stimulatory and signaling domains function to transmit the signaland activate molecules, such as T cells, in response to antigen-binding.The one or more co-stimulatory domains are derived from stimulatorymolecules and/or co-stimulatory molecules, and the signaling domain isderived from a primary signaling domain, such as the CD3 zeta domain. Inembodiments, the signaling domain further includes one or morefunctional signaling domains derived from a co-stimulatory molecule. Inembodiments, the co-stimulatory molecules are cell surface molecules(other than antigens receptors or their ligands) that are required foractivating a cellular response to an antigen.

In embodiments, the co-stimulatory domain includes the intracellulardomain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, or any combination thereof. Inembodiments, the signaling domain includes a CD3 zeta domain derivedfrom a T cell receptor.

The CAR molecules described herein also include a transmembrane domain.The incorporation of a transmembrane domain in the CAR moleculesstabilizes the molecule. In embodiments, the transmembrane domain of theCAR molecules is the transmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of theCAR, there may be incorporated a spacer domain. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to the extracellular domain and/or thecytoplasmic domain on the polypeptide chain. A spacer domain may includeup to 300 amino acids, preferably 10 to 100 amino acids, and mostpreferably 25 to 50 amino acids.

Embodiments relate to a modified cell comprising a binding molecule, anda dominant negative form of an inhibitory immune checkpoint molecule,wherein expression of the dominant negative form of the inhibitoryimmune checkpoint molecule is regulated by an inducible gene expressionsystem. Embodiments relate to a polynucleotide encoding the bindingmolecule and/or the dominant negative form of the inhibitory immunecheckpoint molecule. Embodiments relate to a pharmaceutical compositioncomprising the population of the modified cell. Embodiments relate to akit comprising an effective amount of vector-free nucleic acids encodingthe binding molecule and/or the dominant negative form of the inhibitoryimmune checkpoint molecule to render a population of immune cellsspecific for a tumor antigen expressed on the surface of the cells of asubject. Embodiments relate to a method of eliciting or enhancing T cellresponse, treating a subject in need thereof or enhancing cancertreatment thereof, the method comprising administering an effectiveamount of the composition or the kit to the subject.

Embodiments relate to a pharmaceutical composition comprising apopulation of the modified cells and a population of additional modifiedcells, wherein the modified cells bind a first antigen, and theadditional modified cells bind a second antigen, which is different formthe first antigen. In embodiments, the first antigen is a white bloodcell antigen, and the second antigen is a solid tumor antigen. Inembodiments, the second antigen is a white blood cell antigen, and thefirst antigen is a solid tumor antigen. In embodiments, the white bloodcell antigen is CD19, CD22, CD20, BCMA, CDS, CD7, CD2, CD16, CD56, CD30,CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. Inembodiments, the blood cell antigen is CD19, CD20, CD22, or BCMA. Inembodiments, the solid tumor antigen is tMUC 1, PRLR, CLCA1, MUC12,GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207,SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC-15, SLC6A3,KISS1R, CLDN18.2, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP,MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Rα2, Mesothelin,PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, B7-H3, or EGFR.In embodiments, the solid tumor antigen comprises tumor associated MUC1,ACPP, TSHR, GUCY2C, UPK2, CLDN18.2, PSMA, DPEP3, CXCRS, B7-H3, MUC16,CLDN6, Muc17, PRLR, and FZD10.

Embodiments relate to a method or use of polynucleotide, the methodcomprising providing a viral particle (e.g., AAV, lentivirus or theirvariants) comprising a vector genome, the vector genome comprising thepolynucleotide encoding the binding molecule and/or the dominantnegative form of the inhibitory immune checkpoint molecule, thepolynucleotide operably linked to an expression control elementconferring transcription of the polynucleotides; and administering anamount of the viral particle to a subject such that the polynucleotideis expressed in the subject, where the one or more molecules areoverexpressed in cancer cells, associated with recruitment of immunecells, and/or associated with autoimmunity. In embodiments, the AAVpreparation may include AAV vector particles, empty capsids and hostcell impurities, thereby providing an AAV product substantially free ofAAV empty capsids.

In embodiments, expression of the one or more molecules may be regulatedby an inducible expression system. The inducible expression systemallows for a temporal and spatial controlled activation and/orexpression of genes. For example, Tetracycline-ControlledTranscriptional Activation is a method of inducible gene expressionwhere transcription is reversibly turned on or off in the presence ofthe antibiotic tetracycline or one of its derivatives (e.g.,doxycycline). For example, an inducible suicide gene expression systemallows for a temporal and spatial controlled activation and/orexpression of a suicide gene, which causes a cell to kill itself throughapoptosis. In embodiments, the modified cells comprise a nucleic acidsequence encoding a reverse tetracycline transactivator (rtTA). Inembodiments, expression of the one or more molecules is regulated by thertTA, such that the one or more molecules are expressed in the presenceof tetracycline. In embodiments, a concentration of tetracycline in thecell culture medium is not less than about 2 pg/ml. In embodiments, thetetracycline is selected from the group consisting of tetracycline,demeclocycline, meclocycline, doxycycline, lymecycline, methacycline,minocycline, oxytetracycline, rolitetracycline, and chlortetracycline.In embodiments, the tetracycline is doxycycline. In embodiments, theinducible suicide system is an HSV-TK system or an inducible caspase-9system. In embodiments, the modified cells comprise a nucleic acidsequence encoding a suicide gene, such that when the modified cells arein the presence of a nucleoside analogue in a manner permittingexpression of the suicide gene, to render the nucleoside analoguecytotoxic to the modified cells. In embodiments, the suicide gene isselected from the group consisting of thymidine kinase of herpes simplexvirus, thymidine kinase of varicella zoster virus, and bacterialcytosine deaminase. In embodiments, the suicide gene is thymidine kinaseof herpes simplex virus. In embodiments, the nucleoside analogue isselected from the group consisting of ganciclovir, acyclovir,buciclovir, famciclovir, penciclovir, valciclovir, trifluorothymidine,1-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A,araT 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2′-deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIU,dideoxycytidine, and araC. In embodiments, the nucleoside analogue isganciclovir.

In embodiments, expression of the one or more molecules is regulated byone or more promoters. In embodiments, the polynucleotide comprises apromoter comprising a binding site for a transcription modulator thatmodulates the expression and/or secretion of the one or more moleculesin the cell. For example, the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB. For example, the one or more moleculescomprise at least one cytokine associated with an oxygen-sensitivepolypeptide domain, and the oxygen-sensitive polypeptide domaincomprises HIF VHL binding domain.

In embodiments, the polynucleotide may integrate into the genome of themodified cell and descendants of the modified cell will also express thepolynucleotide, resulting in a stably transfected modified cell. Inembodiments, the modified cell may express the polynucleotide encodingthe CAR but the polynucleotide does not integrate into the genome of themodified cell such that the modified cell expresses the transientlytransfected polynucleotide for a finite period of time (e.g., severaldays), after which the polynucleotide is lost through cell division orother factors. For example, the polynucleotide is present in themodified cell in a recombinant DNA construct, in an mRNA, or in a viralvector, and/or the polynucleotide is an mRNA, which is not integratedinto the genome of the modified cell.

Embodiments relate to a method or use of polynucleotide. The method oruse includes: providing a viral particle (e.g., AAV, lentivirus or theirvariants) comprising a vector genome, the vector genome comprising thepolynucleotide, wherein the polynucleotide is operably linked to anexpression control element conferring transcription of thepolynucleotide; and administering an amount of the viral particle to thesubject such that the polynucleotide is expressed in the subject. Inembodiments, the AAV preparation may include AAV vector particles, emptycapsids and host cell impurities, thereby providing an AAV productsubstantially free of AAV empty capsids. More information of theadministration and preparation of the viral particle may be found at theU.S. Pat. No. 9,840,719 and Milani et al., Sci. Transl. Med. 11,eaav7325 (2019) 22 May 2019, which are incorporated herein by reference.

In embodiments, the bioreactor may be inoculated at a cell density ofapproximately 0.5×10⁶ cells/mL with viability greater than 95%. When thecell density reaches approximately 1.0×10⁶cells/mL, the cells may betransfected with the PEI/DNA complexes (polyplexes) with a PEI to DNAratio of 2:1. At the time of harvest, AAV from the cell culture in thebioreactor may be released using the Triton X-100 method. All solutionsmay be added directly to the bioreactor, and the lysate was centrifugedat 4000×g for 20 min. The supernatant may be stored at −80° C. forfurther processing. AAV may be further purified. For example, AAVsamples (12.3 mL) may be purified by overlaying them on top of series ofstep gradients using 15, 25, 40 and 54% iodixanol concentrationscontaining 1, 5, 7 and 5 mL, respectively. The 15% iodixanolconcentration also contains 1 M NaCI to avoid aggregation of AAV withother cellular proteins and negatively charged nuclear components. Afterthe completion of centrifugation, 5 mL may be withdrawn from 2 mm belowthe 40/54 interface marked before starting the ultracentrifugation at385,000×g for 1 h 45 min in Sorvall T-865 rotor in SorvallUltracentrifuge. The viral vectors may be then quantified. For example,vectors AAV infectivity may be determined by the gene transfer assay(GTA)using GFP as a reporter gene in all cases. AAV infectivity assaywhere sample may be diluted before addition to the cells to have the GFPpositive cells in the range of 2-20% to assure that only single virushas entered the cell for GFP expression. The GFP-positive cells may bequantified by FACS using HEK293 cells in suspension. The AAV may be thenadministrated to a subject. For example, AAV may be diluted in 0.9%sterile NaCI saline solution (supplemented with 0.25% human serumalbumin [HSA]) for infusion in patients and the final volume of infusionwill be calculated based on the patient's weight as 3 mL/kg.

The term “expression or overexpression” refers to the transcriptionand/or translation of a particular nucleotide sequence into a precursoror mature protein, for example, driven by its promoter. “Overexpression”refers to the production of a gene product in transgenic organisms orcells that exceeds levels of production in normal or non-transformedorganisms or cells. As defined herein, the term “expression” refers toexpression or overexpression.

In embodiments, a T cell clone that expresses a TCR with a high affinityfor the target antigen may be isolated. Tumor-infiltrating lymphocytes(TILs) or peripheral blood mononuclear cells (PBMCs) can be cultured inthe presence of antigen-presenting cells (APCs) pulsed with a peptiderepresenting an epitope known to elicit a dominant T cell response whenpresented in the context of a defined HLA allele. High-affinity clonesmay be then selected on the basis of MHC-peptide tetramer stainingand/or the ability to recognize and lyse target cells pulsed with lowtitrated concentrations of cognate peptide antigen. After the clone hasbeen selected, the TCRα and TCRβ chains or TCRγ and TCRδ chains areidentified and isolated by molecular cloning. For example, for TCRα andTCRβ chains, the TCRα and TCRβ gene sequences are then used to generatean expression construct that ideally promotes stable, high-levelexpression of both TCR chains in human T cells. The transductionvehicle, for example, a gammaretrovirus or lentivirus, can then begenerated and tested for functionality (antigen specificity andfunctional avidity) and used to produce a clinical lot of the vector. Analiquot of the final product can then be used to transduce the target Tcell population (generally purified from patient PBMCs), which isexpanded before infusion into the patient.

Various methods may be implemented to obtain genes encodingtumor-reactive TCR. More information is provided in Kershaw et al., ClinTransl Immunology. 2014 May; 3(5): e16. In embodiments, specific TCR canbe derived from spontaneously occurring tumor-specific T cells inpatients. Antigens included in this category include the melanocytedifferentiation antigens MART-1 and gp100, as well as the MAGE antigensand NY-ESO-1, with expression in a broader range of cancers. TCRsspecific for viral-associated malignancies can also be isolated, as longas viral proteins are expressed by transformed cells. Malignancies inthis category include liver and cervical cancer, associated withhepatitis and papilloma viruses, and Epstein-Barr virus-associatedmalignancies. In embodiments, target antigens of the TCR may include CEA(e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for Melanoma),MAGE-A3 (e.g., Melanoma, esophageal and synovial sarcoma), NY-ESO-1(e.g., for Melanoma and sarcoma as well as Multiple myelomas).

In embodiments, preparation and transfusion of tumor infiltratinglymphocytes (TIL) may be implemented by the following. For example,tumor tissue comes from surgical or biopsy specimens, may be obtainedunder aseptic conditions and transported to the cell culture chamber inice box. Necrotic tissue and adipose tissue may be removed. The tumortissue may be cut into small pieces of about 1-3 cubic millimeter.Collagenase, hyaluronidase and DNA enzyme may be added, and digestedovernight at 4° C. Filtering with 0.2 um filter, cells may be separatedand collected by lymphocyte separation fluid, 1500 rpm for 5 min.Expanding the cells with a culture medium comprising PHA,2-mercaptoethanol and CD3 monoclonal antibody, a small dose of IL-2(10-20 IU/ml) may be added to induce activation and proliferation.According to the growth situation, the cell density may be carefullydetected and maintained within the range of 0.5-2*10{circumflex over( )}6/ml under the condition of 37° C. and 5% CO2 for 7-14 days. TILpositive cells have the ability to kill homologous cancer cell may bescreened out by co-culture. The positive cells may be amplified in aserum-free medium containing a high dose of IL2 (5000-6000 IU/ml) untilgreater than 1*10{circumflex over ( )}11 TILs may be obtained. Toadminister TILs, they may be first collected in saline usingcontinuous-flow centrifugation and then filtered through aplatelet-administration set into a volume of 200-300 mL containing 5%albumin and 450 000 IU of IL-2. The TILs may be infused into patientsthrough a central venous catheter over a period of 30-60 minutes. Inembodiments, TILs may be often infused in two to four separate bags; theinfusions may be separated by several hours.

In embodiments, expression of the polynucleotide is regulated ormodulated by a synthetic Notch receptor comprising, from N-terminal toC-terminal and in covalent linkage: a) an extracellular domaincomprising an antibody (e.g., a single-chain Fv (scFv) or a nanobody)that specifically binds to an antigen; b) a Notch regulatory region(NRR) and c) an intracellular domain comprising a transcriptionalactivator comprising a DNA binding domain. In embodiments, the Notchregulatory region comprises a Lin 12-Notch repeat, a heterodimerizationdomain comprising an S2 proteolytic cleavage site and a transmembranedomain comprising an S3 proteolytic cleavage site. The intracellulardomain is heterologous to the Notch regulatory region. In embodiments,the transcriptional activator replaces a naturally-occurringintracellular notch domain, and binding of the antibody to the antigeninduces cleavage at the S2 and S3 proteolytic cleavage sites, therebyreleasing the intracellular domain. The release of the intracellulardomain causes the transcriptional activator to induce expression of thepolynucleotide encoding one or more target proteins in the modifiedcell. In embodiments, the modified cell comprises a polynucleotideencoding the synthetic Notch receptor and a polynucleotide encoding atranscriptional control element that is responsive to thetranscriptional activator and operably linked to the polynucleotideencoding one or more target proteins (e.g., CAR and scFv targeting M2).

“Suicide gene” is a nucleic acid coding for a product, wherein theproduct causes cell death by itself or in the presence of othercompounds. A representative example of such a therapeutic nucleic acid(suicide gene) is one which codes for thymidine kinase of herpes simplexvirus (HSV-TK). Additional examples are thymidine kinase of varicellazoster virus and the bacterial gene cytosine deaminase which can convert5-fluorocytosine to the highly toxic compound 5-fluorouracil.

In embodiment, the modified cell comprises an inducible gene expressionsystem that comprises or is a lac system, a tetracycline system, or agalactose system. In embodiment, the expression of the dominant negativeform of the inhibitory immune checkpoint molecule is regulated by Hif1a,NFAT, FOXP3, and/or NFkB.

In embodiment, the modified cell comprises a polynucleotide encoding thebinding molecule and a dominant negative form of the inhibitory immunecheckpoint molecule or a receptor thereof, wherein expression of thedominant negative form of the inhibitory immune checkpoint molecule isregulated by an inducible gene expression system.

In embodiment, the modified cell comprises a polynucleotide encoding thedominant negative form of the inhibitory immune checkpoint molecule orthe receptor thereof, wherein expression of the dominant negative formof the inhibitory immune checkpoint molecule is regulated by aninducible gene expression system. In embodiments, the modified cellcomprises a sequence listed in Table 7.

In embodiment, the polynucleotide comprises a polynucleotide encoding aNFAT promoter operatively associated with a nucleotide sequence encodingthe inhibitory immune checkpoint molecule or the receptor thereof.

Embodiments relate to a modified cell comprising a polynucleotideencoding a secretable antibody binding SIGLEC-15 and/or encoding adominant negative form of CD44. The antibody binding SIGLEC-15 can be ascFv. Embodiments relate to a modified cell comprising a polynucleotideencoding a secretable scFv binding SIGLEC-15 and/or encoding asecretable scFv binding CD44. In embodiments, the scFv binding SIGLEC-15or CD44 is attached to the membrane of the modified cell as shown inFIG. 1.

Embodiments relate to a fusion protein comprising a scFv bindingSIGLEC-15, a linker, an extracellular domain, a transmembrane domain,and a cytoplasmic domain, wherein the transmembrane domain is selectedfrom a group consist of a transmembrane domain of a receptor of IL15,IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, andIFNβ, and the cytoplasmic domain is selected from a group consist of acytoplasmic domain of receptor of the receptor of IL15, IL2, IL7, IL6,IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and theextracellular domain is selected from a group consist of anextracellular domain of the receptor of IL15, IL2, IL7, IL6, IL12, IL18,IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ. Embodiments relate to afusion protein comprising a scFv binding SIGLEC-15, a linker, atransmembrane domain, and a cytoplasmic domain, wherein thetransmembrane domain is selected from a group consist of a transmembranedomain of a receptor of CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1,and the cytoplasmic domain is selected from a group consist of acytoplasmic domain of receptor of the receptor of CD4, CD8, CD28, CD27,CD25, CD137, PD1 and PDL1. Embodiments relate to a nucleic acid sequenceencoding the fusion protein.

In embodiments, the binding domain is a scFv. In embodiments, the linkis a GS linker. In embodiments, the scFv binds to one of the SEQ ID NOs:85-89, the extracellular domain is or comprises a SEQ ID NO: 36-42, thetransmembrane domain is or comprises one of the SEQ ID NOs: 43-49,and/or the cytoplasmic domain is or comprises one of the SEQ ID NO:50-56; or the scFv binds to one of the SEQ ID NOs: 85-89, the liner isor comprises a SEQ ID NO: 83 and 84, the transmembrane domain is orcomprises one of the SEQ ID NOs: 59-63, and/or the cytoplasmic domain isor comprises one of the SEQ ID NO: 64-70.

Embodiments relate to a nucleic acid sequence encoding a dominantnegative form of SIGLEC-15 receptor on T cells or the dominant negativeform of SIGLEC-15 receptor and CAR or modified TCR, wherein the modifiedSIGLEC-15 receptor and the CAR/TCR are expressed as gene products thatare separate polypeptides. Embodiments relate to a nucleic acid sequenceencoding a dominant negative form of SIGLEC-15 receptor on T cells(e.g., CD44) or the dominant negative form of CD44 and CAR or modifiedTCR, wherein the modified CD44 and the CAR/TCR are expressed as geneproducts that are separate polypeptides. Dominant negative mutationshave an altered gene product that acts antagonistically to the wild-typeallele. These mutations usually result in an altered molecular function(often inactive) and are characterized by a dominant or semi-dominantphenotype.

In embodiments, the dominant negative form of CD44 comprises asubstitution or deletion as compared to a wild-type CD44 intracellulardomain. SIGLEC-15 is a member of the SIGLEC gene family and has atypical sialic acid-binding immunoglobulin-type lectin structure, whichplays a very important role in osteoclast differentiation and boneremodeling [1-4]. Expression of SIGLEC-15 is typically restricted tomyeloid cells and is up-regulated in many human cancers. SIGLEC-15 is animmunosuppressive molecule that acts primarily on the tumormicroenvironment and has no correlation with the PD-L1/PD-1 pathway.CD44 may be a potential functional ligand for SIGLEC-15 and is involvedin the activation of T cells. The dominant negative receptor isconstructed, that is, the extracellular segment of CD44 is retained, andits transmembrane or intracellular segment is modified to bind toSIGLEC-15, but the inhibitory signal cannot be transmitted through itscytoplasmic domain such as to enhance the anti-tumor activity of CAR-Tcells.

Embodiments relate to a modified cell comprises the fusion protein orthe nucleic acid sequence. Embodiments relate to a modified cellcomprises a dominant negative form of CD44. In embodiments, the dominantnegative form of CD44 is or comprises one of the SEQ ID NOS: 94-96. Inembodiments, the modified cell is a T cell, NK cell, or dendritic cells.

Embodiments relate to a pharmaceutical composition comprising thepopulation of the modified cells. Embodiments relate to a method ofcause T cell response in a subject in need thereof and/or treating atumor of the subject, the method comprising administering an effectiveamount of the composition to the subject.

Embodiments relate to a method of causing T cell response, treating asubject having cancer, enhancing cellular treatment on a subject havingcancer, or inhibiting growth of tumor cells, the method comprising:administering an effective amount of modified cells comprising a bindingmolecule to the subject; and administering an effective amount of aninhibitor of an inhibitory immune checkpoint molecule (e.g., moleculesassociated with SIGLEC-15 and CD44 pathway) or a receptor thereof inresponse to a predetermined condition. In embodiments, the methodfurther comprises intruding into cells with a plurality of nucleic acidsequences encoding the binding molecule to obtain the modified cells. Inembodiments, the method further comprises monitoring cytokine releasesof the subject in response to the CAR infusion; and wherein theadministering the effective amount of the inhibitor of the inhibitoryimmune checkpoint molecule or the receptor thereof in response to thepredetermined condition comprises administering the effective amount ofthe inhibitor of the inhibitory immune checkpoint molecule or thereceptor thereof in response to determination that a level of one ormore cytokines increases and/or reaches a peak level. In embodiments,the method further comprises terminating the administering of theinhibitor in response to determination that the level drops to a baselevel. In embodiments, the cytokine may include IL6 and/or IFNγ, asshown in FIG. 11.

In embodiments, the modified cell comprises a CAR. In embodiments, theCAR comprises an extracellular domain, a transmembrane domain, and anintracellular domain (e.g., 41-BB and CD3 zeta domain), theextracellular domain binds an antigen. In embodiments, the intracellulardomain comprises a costimulatory signaling region that comprises anintracellular domain of a costimulatory molecule selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and any combination thereof. In embodiments, the antigenis Epidermal growth factor receptor (EGFR), Variant III of the epidermalgrowth factor receptor (EGFRvIII), Human epidermal growth factorreceptor 2 (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen(PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX(CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125),Cluster of differentiation 133 (CD133), Fibroblast activation protein(FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folatereceptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3,CD5, B-Cell Maturation Antigen (BCMA), or CD4.

TABLE 2 SEQ ID NO: Identity 1 SP 2 Hinge & transmembrane domain 3Co-stimulatory region 4 CD3-zeta 5 scFV Humanized CD19 6 scFV CD19 7scFv FZD10 8 scFv TSHR 9 scFv PRLR 10 scFv Muc 17 11 scFv GUCY2C 12 scFvCD207 13 Prolactin (ligand) 14 scFv CD3 15 scFv CD4 16 scFv CD4-2 17scFv CD5 18 WTCD3zeta 19 WTCD3zeta-BCMACAR full length 20 BCMACAR 21MUC1CAR 22 m19CAR-IRES-MUC1CAR 23 hCD19CAR-IRES-MUC1CAR 24hCD22CAR-IRES-MUC1CAR 25 BCMACAR-IRES-MUC1CAR 26 mCD19CAR-2A-MUC1CAR 27hCD19CAR-2A-MUC1CAR 28 hCD22CAR-2A-MUC1CAR 29 BCMA-2A-MUC1CAR 30 Tumorassociated MUC1 scFv 1 31 Tumor associated MUC1 scFv-1 VH 32 Tumorassociated MUC1 scFv-1 VL 33 Tumor associated MUC1 scFv 2 34 Tumorassociated MUC1 scFv2 VH 35 Tumor associated MUC1 scFv2 VL 36 ED IL2receptor 37 ED IL6 receptor 38 ED IL7 receptor 39 ED IL12 receptor 40 EDIL15 receptor 41 ED IL21 receptor 42 ED IL23 receptor 43 TM IL2 receptor44 TM IL6 receptor 45 TM IL7 receptor 46 TM IL12 receptor 47 TM IL15receptor 48 TM IL21 receptor 49 TM IL23 receptor 50 CD IL2 receptor 51CD IL6 receptor 52 CD IL7 receptor 53 CD IL12 receptor 54 CD IL15receptor 55 CD IL21 receptor 56 CD IL23 receptor 57 TM CD4 58 TM CD8 59TM CD27 60 TM CD28 61 TM CD137 62 TM PD1 63 TM PDL1 64 CD CD4 65 CD CD866 CD CD27 67 CD CD28 68 CD CD137 69 CD PD1 70 CD PDL1 71 IL2 72 IL6 73IL7 74 IL12 75 IL15 76 IL21 77 IL23 78 IL33 79 TNFα 80 TNFβ 81 HifVHL-interaction domain: Hif amino acid 344-417 82 Hif amino acid 380-60383 GS linker sequence 84 EA linker sequence 85 SIGLEC-15 antigen 1 86SIGLEC-15 antigen 2 87 SIGLEC-15 antigen 3 88 SIGLEC-15 antigen 4 89SIGLEC-15 antigen 5 90 SIGLEC-15 antigen 6 91 SIGLEC-15 antigen 7 92SIGLEC-15 antigen 8 93 CD44 VVT aa 94 Truncated CD44 aa 95 CD44 + CD8αaa 96 CD44 point mutant aa 97 h-Notch transmembrane 98 m-Notchtransmembrane 99 OX40 extracellular 100 CD40 extracelluar 101 41-BBextracellular 102 Her2 extracellular 103 GITR extracellular 104 CD28extracellular 105 ICOS extracellular 106 EGFR extracellular 107 CD27extracellular 108 OPA1 109 MKN1 110 MKN2 111 DNM1L 112 RUNX3 113 EOMES114 SIGLEC-15-P2A-EGFP 115 Anti-SIGLEC-15 scFv1 116 Anti-SIGLEC-15 scFv2117 DNM1L 118 mCD19-P2A-DNM1L 119 P2A-DNM1L Note: EM: ExtracellularDomain; TM: Transmembrane Domain; CD: Cytoplasmic Domain

Embodiments relate to a fusion protein comprising an extracellulardomain, a transmembrane domain, and a cytoplasmic domain, theextracellular domain being or comprising the extracellular domain of atleast one of OX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR,the transmembrane domain being or comprising the transmembrane domain ofan inducible molecule, and the cytoplasmic domain being or comprising atleast one of OPA1, MKN1, MKN2, DNM1L, runx3, EOMES, TCF1, LEF1, Runx1,STAT3, TRAF6, ID3.

Embodiments relate to a fusion protein comprising an extracellulardomain, a transmembrane domain, and a cytoplasmic domain, thetransmembrane domain being or comprising the transmembrane domain ofNotch. More information about Notch and its components may be found inU.S. Pat. No. 9,670,281, which incorporated herein by reference.

In embodiments, the extracellular domain comprises an extracellulardomain of a receptor, an antibody, or a ligand. In embodiments, thereceptor is a costimulatory molecule. In embodiments, the receptor isOX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR. In embodiments,the ligand corresponds to the receptor. In embodiments, the receptor isa cytokine receptor, and the ligand is a cytokine. In embodiments, theantibody is an scFv targeting a tumor antigen. In embodiments, thecytoplasmic domain comprises a mitochondrial protein or a transcriptionfactor. In embodiments, the cytoplasmic domain comprises at least one ofOPA1, MKN1, MKN2, DNM1L, runx3, EOMES, TCF1, LEF1, Runx1, STAT3, TRAF6,and ID3. In embodiments, the cytoplasmic domain comprises at least oneof a protein listed in Table 3.

In embodiments, the extracellular domain corresponds to an agent, andthe transmembrane domain of Notch comprises a TACE cleavage site and aγ-secretase cleavage site such that binding of an extracellular domainto the agent induces cleavage at the TACE cleavage site and theγ-secretase cleavage site, thereby releasing the cytoplasmic domain. Inembodiments, the agent is the receptor, and the extracellular domaincomprises the ligand of the receptor. In embodiments, the extracellulardomain comprises an extracellular domain of the receptor, and the agentis a ligand corresponding to the receptor. In embodiments, the agent isthe tumor antigen, and the extracellular domain comprises an antibodyagainst the tumor antigen.

In embodiments, the extracellular domain comprises at least one of SEQID NO: 38-46, the transmembrane domain comprises at least one of SEQ IDNO: 36 and 37, and the cytoplasmic domain comprises at least one of SEQID NO: 47-52.

Embodiments relate to a nucleic acid sequence encoding the fusionprotein. Embodiments relate to a modified cell comprises a fusionprotein described herein, for example one of embodiments 1-16 or thenucleic acid encoding a fusion protein described herein (one ofembodiments 1-16). Embodiments relate to a pharmaceutical compositioncomprising the population of the modified cells. Embodiments relate to amethod of cause T cell response in a subject in need thereof and/ortreating a tumor of the subject, the method comprising administering aneffective amount of the composition.

In embodiments, the modified cell comprises a CAR. In embodiments, theCAR comprises an extracellular domain, a transmembrane domain, and anintracellular domain, and the extracellular domain binds an antigen. Inembodiments, the pharmaceutical composition, the modified cell, or themethod of promoting a T cell response described herein include theintracellular domain comprises a costimulatory signaling region thatcomprises an intracellular domain of a costimulatory molecule selectedfrom the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and any combination thereof. In embodiments, the antigenis Epidermal growth factor receptor (EGFR), Variant III of the epidermalgrowth factor receptor (EGFRvIII), Human epidermal growth factorreceptor 2 (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen(PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX(CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125),Cluster of differentiation 133 (CD133), Fibroblast activation protein(FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folatereceptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3,CDS, B-Cell Maturation Antigen (BCMA), or CD4. In embodiments, the cellis a T cell, macrophage, dendritic cell, or NK cell.

Embodiments relate to a fusion protein that can be activated by agonistantibodies to perform the specific desired functions. In anoverexpressed fusion protein, the extracellular domain comprise aprotein sequence that can be bound by an agonist antibody (that is, anantibody that binds the extracellular domain of the fusion protein), andthe intracellular is the intracellular domain of another transmembraneprotein. When using the agonist antibody, the extracellular segment iscross-linked by the agonist antibody, and the intracellular segment ofthe fusion protein forms a cluster that activates the downstreampathway. Examples of the fusion protein and the agonist antibody areshown in FIG. 12. An “agonist antibody,” as used herein, is an antibodywhich activates a biological activity of the antigen it binds. Inembodiments, during agonist antibody activation, partial dissociation ofantibodies allows the antigen-binding fragment (Fab) arms of a singleantibody to interact with more than two receptors in a dynamic fashion,resulting in the recruitment of multiple receptor monomers into areceptor oligomer where signaling activation can be triggered. Inembodiments, antigen-presenting cells (APCs) acts as a scaffold tocrosslink agonist antibody bound to a receptor (e.g., co stimulatoryreceptor), leading to receptor supercluster formation and increasedagonist signaling. Some proteins (such as the proteins of the CD28family and the TNF receptor family) have an agonist antibody for tumortherapy. More information about agonist antibodies and co-stimulatorymolecules may be found at Nature Reviews Drug Discovery volume 17, pages509-527 (2018), which is incorporated herein by reference in itsentirety.

TABLE 3 Group 1 Group 1 Group 1 Group 1 Group 2 Group 3 Group 4 LEF1CXCR3 FRC T-bet PI3K ezh2 PDCD1 Gfi1 Flt3 FAO GATA3 Akt kiaa1324 PDL1δEF1/ZEB Kit Hif-1a Blimp-1 mTOR fn1 PDL2 Ikaros IL-7R Hif-2a Myc WntITGA9 CTLA4 KLF2 IL-4R Vhl HIF1 Notch1 SMDPD3A LRBA HEB IL-9R PHD1 c-MybNotch2 ITPRIPL2 LAG3 MAZR PDGFRb PHD2 members of the Activin PRSS23 Tim3E2A/HEB family Tox gc (Il2rg PHD3 members of the BMP CLECL1 BILA Ikarosfamily PU.1 IL-2Ra FIH1 Ets-family TGFb TBX21 CD160 (CD25) transcriptionfactor PU.1 HES1,2,3 IL-2Rb TET2 Runx1 IL7 IKZF2 2B4 Sox4 PDGFRa TET1GATA-2 IL7Ra EOMES Foxp3 E12, Pdgfa TET3 TCF-1 (Tcf7) IL12 PRDM1 ccr4E2-2 Pdgfb DNMT3 RBPSuh IL15 BTLA PVRIG E47, Pdgfc Id3 IL6 CD244 CD16BMEF2 Pdgfd Id2 IL2 KL6 SIVA1 Nur77/Nor1 TGF-b1 Runx1 MAPK JUNB CD33Ncoa4 TGF-b2 Runx3 AMPK FOSB LAGLS9 Basp2 TGF-b3 EBF NF-Kb FAM13A CD122Pitx1 BMP2 Pax5 NF-AT BATF3 IDO1 Prdm16 BMP4 FOG-1 AP-1 KLRC1 IDO2 NdnBMP7 GATA-3 PI-3 KLRC2 CD45 kinase, Akt/PKB, and Ras/MAP Irf6ActivinbetaA GATA-2 CCR7 ZNF704 CVPLBL Dach1 ALK-5 GATA-1 STAT1 CTHRC1TNFAIP8L2 Nr4a2 BMPR-1A/B Groucho/TLE/ STAT2 FAXC DNMT3A Grg familyproteins or Sin3A Hoxa5 BMPR-II Helios STAT3 EGR1 CEACAM-1 Hoxb5 TLRNFAT STAT4 RBM47 RUNX3 FoxN1 MyD88 Bcl-6 STAT5 ENTPD1 LEXM Gli1,2,3 SHHBCL-6b (BAZF) glut1 SUV39H1 PILRA Smoothen HIF1 AKAP5 PTNNS1L3 Ptch1c-Myc β-catenin Fegr3a Fu IRF4 PI3K Nat8 Su(fu) NF-kb GLUL ccl9Wnt1,3,4,5b,10b ThPOK FAXC HCK Smad Oct3/4 TREM2 CXCL10 Nanog LNGM CCL6CXCL9 Sox2 NUDT16 CD36 INFa,b KLF4 IGF1 CXCR3 FOXO1 CTSS CCL5 TSC1 GZMCCCR5 OxPhos BATF CCR7 Zbtb32 CXCL2 SOCS1 E2F2 TNFAIP8L 3 SOCS2 SMARTIL-1b SOCS3 NCOR IL-1a SOCS4 TRPV1 SOCS5 TRPV2 SOCS6 TRPV3 SOCS7 TRPV4CIS Rgs1 PLSCR1 ITGB1 ITGB2 C3AR1 ITGA3 ITGA5 ITGAL CARD11 CD83

The present disclosure is further described by reference to thefollowing exemplary embodiments and examples. These exemplaryembodiments and examples are provided for purposes of illustration onlyand are not intended to be limiting unless otherwise specified. Thus,the present disclosure should in no way be construed as being limited tothe following exemplary embodiments and examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Exemplary Embodiments

The following are exemplary embodiments:

-   1. A fusion protein comprising an extracellular domain, a    transmembrane domain, and a cytoplasmic domain, the extracellular    domain being or comprising an extracellular domain of at least one    of OX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR, the    transmembrane domain being or comprising a transmembrane domain of    an inducible molecule, and the cytoplasmic domain being or    comprising at least one of OPA1, MKN1, MKN2, DNM1L, runx3, EOMES,    TCF1, LEF1, Runx1, STAT3, TRAF6, or ID3.-   2. A fusion protein comprising an extracellular domain, a    transmembrane domain, and a cytoplasmic domain, the transmembrane    domain being or comprising the transmembrane domain of Notch.-   3. The fusion protein of embodiment 2, wherein the extracellular    domain comprises an extracellular domain of a receptor, an antibody,    or a ligand.-   4. The fusion protein of embodiment 3, wherein the receptor is a    costimulatory molecule.-   5. The fusion protein of embodiment 3, wherein the receptor is OX40,    CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR.-   6. The fusion protein of embodiment 3, wherein the ligand    corresponds to a receptor.-   7. The fusion protein of embodiment 3, wherein the receptor is a    cytokine receptor, and the ligand is a cytokine.-   8. The fusion protein of embodiment 3, wherein the antibody is a    scFv that targets a tumor antigen.-   9. The fusion protein of one of embodiments 2-8, wherein the    cytoplasmic domain comprises a mitochondrial protein or a    transcription factor.-   10. The fusion protein of embodiment 9, wherein the cytoplasmic    domain comprises at least one of OPA1, MKN1, MKN2, DNM1L, runx3,    EOMES, TCF1, LEF1, Runx1, STAT3, TRAF6, or ID3.-   11. The fusion protein of embodiment 9, wherein the cytoplasmic    domain comprises at least one of a protein listed in Table 3.-   12. The fusion protein of one of embodiments 2-11, wherein the    extracellular domain corresponds, to, interacts or associates with,    and/or binds an agent, and the transmembrane domain of Notch    comprises a TACE cleavage site and a y-secretase cleavage site such    that binding of an extracellular domain to the agent induces    cleavage at the TACE cleavage site and the y-secretase cleavage    site, thereby releasing the cytoplasmic domain.-   13. The fusion protein of embodiment 12, wherein the agent is a    receptor, and the extracellular domain comprises a ligand of the    receptor.-   14. The fusion protein of embodiment 12, wherein the extracellular    domain comprises an extracellular domain of a receptor, and the    agent is a ligand of the receptor.-   15. The fusion protein of embodiment 12, wherein the agent is a    tumor antigen, and the extracellular domain comprises an antibody    against the tumor antigen.-   16. The fusion protein of embodiment 12, wherein the extracellular    domain comprises an amino acid sequence of at least one of SEQ ID    NO: 99-106, the transmembrane domain comprises an amino acid    sequence of at least one of SEQ ID NO: 97 and 98, and the    cytoplasmic domain comprises an amino acid sequence of at least one    of SEQ ID NO: 108-113.-   17. A nucleic acid encoding the fusion protein of one of embodiments    1-16.-   18 A modified cell comprising the fusion protein of one of    embodiments 1-16 or the nucleic acid sequence of embodiment 17.-   19. A pharmaceutical composition comprising a population of the    modified cells of embodiment 18.-   20. A method of causing or promoting a T cell response in a subject    in need thereof and/or treating a tumor of the subject, the method    comprising administering an effective amount of the composition of    embodiment 19 to the subject.-   21. The pharmaceutical composition, the modified cell, and the    method of one of embodiments 17-20, wherein the modified cell    comprises a CAR.-   22. The pharmaceutical composition, the modified cell, and the    method of one of embodiments 17-20, wherein the CAR comprises an    extracellular domain, a transmembrane domain, and an intracellular    domain, and the extracellular domain binds an antigen.-   23. The pharmaceutical composition, the modified cell, and the    method of embodiment 23, wherein the intracellular domain comprises    a costimulatory signaling region that comprises an intracellular    domain of a costimulatory molecule selected from the group    consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,    lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,    NKG2C, B7-H3, and a combination thereof.-   24. The pharmaceutical composition, the modified cell, and the    method of embodiment 23, wherein the antigen is Epidermal growth    factor receptor (EGFR), Variant III of the epidermal growth factor    receptor (EGFRvIII), Human epidermal growth factor receptor 2    (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen    (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),    Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase    IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125    (CA125), Cluster of differentiation 133 (CD133), Fibroblast    activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1    (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,    GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4.-   25. The pharmaceutical composition, the modified cell, and the    method of embodiments 18-24, wherein the cell is a T cell,    macrophage, dendritic cell, or NK cell.-   26. A fusion protein comprising an scFv binding SIGLEC-15, a linker,    an extracellular domain, a transmembrane domain, and a cytoplasmic    domain, wherein the transmembrane domain is selected from a group    consisting of a transmembrane domain of a receptor of IL15, IL2,    IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ,    and the cytoplasmic domain is selected from a group consisting of a    cytoplasmic domain of receptor of the receptor of IL15, IL2, IL7,    IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and    the extracellular domain is selected from a group consisting of an    extracellular domain of the receptor of IL15, IL2, IL7, IL6, IL12,    IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ.-   27. A fusion protein comprising an scFv binding SIGLEC-15, a linker,    a transmembrane domain, and a cytoplasmic domain, wherein the    transmembrane domain is selected from a group consisting of a    transmembrane domain of a receptor of CD4, CD8, CD28, CD27, CD25,    CD137, PD1, and PDL1, and the cytoplasmic domain is selected from a    group consisting of a cytoplasmic domain of receptor of the receptor    of CD4, CD8, CD28, CD27, CD25, CD137, PD1, and PDL1.-   28. The fusion protein of one of embodiments 26 and 27, wherein the    binding domain is an scFv.-   29. The fusion protein of one of embodiments 26-28, wherein the link    is a GS linker.-   30. The fusion protein of one of embodiments 26-28, wherein the scFv    binds one of the amino acid sequences of SEQ ID NOs: 85-89, the    extracellular domain is or comprises one of amino acid sequences of    SEQ ID NO: 36-42, the transmembrane domain is or comprises one of    the amino acid sequences of SEQ ID NOs: 43-49, and/or the    cytoplasmic domain is or comprises one of the amino acid sequences    of SEQ ID NO: 50-56; or the scFv binds to one of the amino acid    sequences of SEQ ID NOs: 85-89, the linker is or comprises SEQ ID    NO: 83 or 84, the transmembrane domain is or comprises one of amino    acid sequences of SEQ ID NOs: 59-63, and/or the cytoplasmic domain    is or comprises one of the amino acid sequences of SEQ ID NOs:    64-70.-   31. A nucleic acid sequence encoding the fusion protein of one of    embodiments 26-30.-   32. A nucleic acid sequence encoding a dominant negative form of    SIGLEC-15 receptor on T cells or the dominant negative form of    SIGLEC-15 receptor and CAR or modified TCR, wherein the modified    SIGLEC-15 receptor and the CAR/TCR are expressed as gene products    that are separate polypeptides.-   33. A nucleic acid sequence encoding a dominant negative form of    SIGLEC-15 receptor on T cells (e.g., CD44) or the dominant negative    form of CD44 and CAR or modified TCR, wherein the modified CD44 and    the CAR/TCR are expressed as gene products that are separate    polypeptides.-   34. The nucleic acid sequence of embodiment 33, wherein the dominant    negative form of CD44 comprises a substitution or deletion in its    intracellular domain as compared to a wild-type CD44.-   35 A modified cell comprising the fusion protein of one of    embodiments 1-6 or the nucleic acid sequence encoding one of the    fusion proteins of embodiments 9-10.-   36. A modified cell comprising a dominant negative form of CD44.-   37. The modified cell of embodiment 35 or 36, wherein the dominant    negative form of CD44 is or comprises one of the amino acid sequence    of SEQ ID NOs: 94-96.-   38. The modified cell of one of embodiments 35-37, wherein the    modified cell is a T cell, NK cell, or dendritic cells.-   39. A pharmaceutical composition comprising the population of the    modified cells of embodiment 38.-   40. A method of causing or promoting a T cell response in a subject    in need thereof and/or treating a tumor of the subject, the method    comprising administering an effective amount of the composition of    embodiment 39 to the subject.-   41. The pharmaceutical composition, the modified cell, and the    method of one of embodiments 35-40, wherein the modified cell    comprises a CAR.-   42. The pharmaceutical composition, the modified cell, and the    method of embodiment 41, wherein the CAR comprises an extracellular    domain, a transmembrane domain, and an intracellular domain (e.g.,    41-BB and CD3 zeta domain), the extracellular domain binds an    antigen.-   43. The pharmaceutical composition, the modified cell, and the    method of embodiment 41 or 42, wherein the intracellular domain    comprises a costimulatory signaling region that comprises an    intracellular domain of a costimulatory molecule selected from the    group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,    lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,    NKG2C, B7-H3, and a combination thereof.-   44. The pharmaceutical composition, the modified cell, and the    method of embodiment 41 or 42, wherein the antigen is Epidermal    growth factor receptor (EGFR), Variant III of the epidermal growth    factor receptor (EGFRvIII), Human epidermal growth factor receptor 2    (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen    (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),    Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase    IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125    (CA125), Cluster of differentiation 133 (CD133), Fibroblast    activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1    (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,    GUCY2C, CD207, CD3, CDS, B-Cell Maturation Antigen (BCMA), or CD4.-   45. A recombinant antigen-binding protein or fragment thereof    comprising the amino acid sequence of SEQ ID NO: 115 or 116.-   46. The recombinant antigen-binding protein of embodiment 45,    wherein said protein is an antibody.-   47. The recombinant antigen-binding protein of embodiment 46,    wherein the antibody is a human antibody.-   48. The recombinant antigen-binding protein of embodiment 46,    wherein said antibody or antigen-binding fragment thereof is intact    Ig, Fab, F(ab′)₂, Fv, or scFv.-   49. The antigen-binding protein of embodiment 45, wherein said    antigen-binding protein is a SIGLEC-15 agonist.-   50. The antigen-binding protein of embodiment 45, wherein said    antigen-binding protein is a SIGLEC-15 antagonist.-   51. The antigen-binding protein of embodiment 45, wherein said    antigen-binding protein is a chimeric antigen receptor (CAR).-   52. A nucleic acid encoding an antigen-binding protein of embodiment    45 or at least one of the amino acid sequences of SEQ ID NOS: 115,    116, 94-96, and 100.-   53. A vector comprising the nucleic acid of embodiment 52.-   54. A modified cell comprising an antigen-binding protein of    embodiment 45.-   55. A modified cell comprising a nucleic acid of embodiment 52.-   56. A modified cell comprising a vector of embodiment 53.-   57. An antigen-binding protein of embodiment 45 conjugated to a    therapeutic agent.-   58. The antigen-binding protein of embodiment 57, wherein said    therapeutic agent is a drug, toxin, radioisotope, protein, or    peptide.-   59. A pharmaceutical composition comprising an antigen-binding    protein of embodiment 45.-   60. A pharmaceutical composition comprising a nucleic acid of    embodiment 52.-   61. A pharmaceutical composition comprising a vector of embodiment    53.-   62. A pharmaceutical composition comprising a cell that expresses an    antigen-binding protein of embodiment 45.-   63. A method of increasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of    an antigen-binding protein or an antigen-binding fragment thereof of    embodiment 45 or a nucleic acid that encodes the antigen-binding    protein or an antigen-binding fragment thereof of embodiment 45.-   64. The method of embodiment 63, wherein the antigen-binding protein    or antigen-binding fragment thereof inhibits, reduces, modulates or    abolishes signal transduction mediated by SIGLEC-15.-   65. A method for treatment of a subject having a SIGLEC-15 positive    disease, comprising administering to the subject a therapeutically    effective amount of an antigen-binding protein or an antigen-binding    fragment thereof of embodiment 45 or a nucleic acid that encodes the    antigen-binding protein or an antigen-binding fragment thereof of    embodiment 45.-   66. The method of embodiment 65, wherein said antigen-binding    protein or antigen-binding fragment thereof is a conjugate having a    cytotoxic moiety linked thereto.-   67. The method of embodiment 65, wherein the SIGLEC-15 positive    disease is cancer.-   68. A vector comprising a nucleic acid encoding a recombinant    anti-SIGLEC-15 antigen-binding protein of embodiment 45 and a    nucleic acid encoding a chimeric antigen receptor, wherein said    recombinant anti-SIGLEC-15 antigen-binding protein is not identical    to said chimeric antigen receptor.-   69. A modified cell comprising the vector of embodiment 68.-   68. A modified cell comprising a nucleic acid encoding a recombinant    anti-SIGLEC-15 antigen-binding protein of embodiment 45 and a    nucleic acid encoding a chimeric antigen receptor, wherein said    recombinant anti-SIGLEC-15 antigen-binding protein is not identical    to said chimeric antigen receptor.-   70. A modified cell comprising a recombinant SIGLEC-15    antigen-binding protein of embodiment 45 and a chimeric antigen    receptor, wherein said recombinant anti-SIGLEC-15 antigen-binding    protein is not identical to said chimeric antigen receptor.-   71. A vector or a cell comprising a vector that encodes a SIGLEC-15    antigen-binding protein of embodiment 45, wherein the recombinant    anti-SIGLEC-15 antigen-binding protein is an antibody.-   72. A vector or a cell of embodiment 71, wherein the recombinant    SIGLEC-15 antigen-binding protein is a human antibody.-   73. A vector or a cell of embodiment 71, wherein the recombinant    SIGLEC-15 antigen-binding protein is an intact Ig, Fab, F(ab′)2, Fv,    or scFv.-   74. A vector or cell of embodiment 71, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist.-   75. A vector or cell of embodiment 71, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 antagonist.-   76. A vector or cell of embodiment 71, wherein the recombinant    anti-SIGLEC-15 antigen-binding protein is a secretable protein.-   77. A vector or cell of embodiment 71, wherein the chimeric antigen    receptor specifically binds to CD-19.-   78. A vector or cell of embodiment 71, wherein the chimeric antigen    receptor can be inserted in a human T cell membrane.-   79. The modified cell of any suitable preceding embodiments, wherein    the modified cell is a T cell, NK cell, and/or DC.-   80. A pharmaceutical composition comprising a vector or a cell of    any of embodiments 70-79.-   81. The pharmaceutical composition of embodiment 80 further    comprising a pharmaceutically acceptable carrier.-   82. A method of increasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of a    vector or cell of any of embodiments 70-79, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 antagonist.-   83. A method of increasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of a    pharmaceutical composition comprising a vector or a cell of any of    embodiments 70-79, wherein the recombinant SIGLEC-15 antigen-binding    protein is a SIGLEC-15 antagonist.-   84. The method of embodiment 83, wherein the recombinant SIGLEC-15    antigen-binding protein inhibits, reduces, modulates or abolishes    signal transduction mediated by SIGLEC-15.-   85. A method of decreasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of a    vector or cell of any of embodiments 70-79, or a pharmaceutical    composition of embodiment 80 or 81, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist.-   86. A method of decreasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of a    vector or cell of any of embodiments 70-79, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist.-   87. A method of decreasing a T cell response in a subject comprising    administering to the subject a therapeutically effective amount of a    pharmaceutical composition of embodiment 80, wherein the recombinant    SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist.-   88. A method for treatment of a subject having a PD1-positive    disease, comprising administering to the subject a therapeutically    effective amount of a vector the encodes the or cell of any one of    any of embodiments 70-79, or a pharmaceutical composition of    embodiment 80 or 81.-   89. A method for treatment of a subject having a SIGLEC-15-positive    disease, comprising transducing at least one T cell of the subject    with a nucleic acid encoding a recombinant anti-SIGLEC-15    antigen-binding protein and a nucleic acid encoding a chimeric    antigen receptor, wherein said recombinant anti-SIGLEC-15    antigen-binding protein is not identical to said chimeric antigen    receptor.-   90. The method of embodiment 89, wherein the chimeric antigen    receptor does not specifically bind to SIGLEC-15.-   91. The method of embodiment 89, wherein the SIGLEC-15 positive    disease is cancer.-   92. A vector or a cell comprising a vector that encodes an    antigen-binding protein of any of embodiments 70-79, wherein at    least one of the anti-SIGLEC-15 antigen-binding protein and chimeric    antigen receptor is conjugated to a therapeutic agent.-   93. The vector or the cell of embodiment 92, wherein said    therapeutic agent is a drug, toxin, radioisotope, protein, or    peptide.-   94. The modified cell or the method of any suitable preceding    embodiments, wherein the modified cell is a T cell, NK cell, or    dendritic cell.-   95. A modified cell comprising a polynucleotide encoding a binding    molecule, and a polynucleotide encoding an anti-SIGLEC-15    antigen-binding.-   96. The modified cell of embodiment 95, wherein the anti-SIGLEC-15    antigen-binding protein is a secretable protein.-   97. The modified cell of embodiment 95, wherein the anti-SIGLEC-15    antigen-binding protein is a secretable scFv.-   98. The modified cell of embodiment 95, wherein the modified cell    comprises a dominant negative form of PD-1.-   99. The modified cell of any one of the preceding embodiments,    wherein the cell or modified cell is a T cell derived from a healthy    donor or a subject having cancer.-   100. The modified cell of any of preceding embodiments, wherein the    modified T cell comprises a dominant negative form of a receptor    associated with CD44.-   101. The modified cell of embodiment 100, wherein the modified CD44    lacks a functional CD44 intracellular domain for CD44 signal    transduction, interferes with a pathway between CD44 of a human T    cell of the human cells and SIGLEC-15 of a certain cell, comprises    or is a CD44 extracellular domain or a CD44 transmembrane domain or    a combination thereof, comprises a modified CD44 intracellular    domain comprising a substitution or deletion as compared to a    wild-type CD44 intracellular domain, or comprises or is a soluble    receptor comprising a CD44 extracellular domain that binds to    SIGLEC-15 of a certain cell.-   102. The modified cell of embodiment 101, wherein an inhibitory    effect of SIGLEC-15 on cytokine production of the human T cells of    the population is less than an inhibitory effect of SIGLEC-15 on    cytokine production of human T cells that do not comprise at least a    part of the nucleic acid sequence that encodes the modified CD44.-   103. The modified cell of any of preceding embodiments, wherein the    modified cell is engineered to express and secrete a therapeutic    agent such as a cytokine.-   104. The modified cell of embodiment 103, wherein the therapeutic    agent is or comprises IFN-γ.-   105. The modified cell of embodiment 103, wherein the therapeutic    agent is or comprises at least one of IL-6 or IFN-γ, IL-17, and    CCL19.-   106. The modified cell of embodiment 103, wherein the therapeutic    agent is or comprises IL-15 or IL-12, or a combination thereof.-   107. The modified cell of embodiment 103, wherein the therapeutic    agent is or comprises a recombinant or native cytokine.-   108. The modified cell of embodiment 103, wherein the therapeutic    agent comprises an FC fusion protein associated with a small    protein.-   109. The modified cell of embodiment 108, wherein the small protein    is or comprises IL-12, IL-15, IL-6 or IFN-γ.-   110. The modified cell of embodiment 103, wherein the therapeutic    agent is regulated by Hif1a, NFAT, FOXP3, and/or NFkB.-   111. The modified cell of embodiment 108, wherein the small protein    or the therapeutic agent is or comprises two or more recombinant or    native cytokines are collected via 2A or /IRES component.-   112. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the modified T cell comprises a first    targeting vector and a second targeting vector, the first targeting    vector comprising a nucleic acid sequence encoding a CAR binding a    blood antigen and the therapeutic agent, and the second targeting    vector comprises a nucleic acid sequence encoding a CAR biding solid    tumor antigen and a dominant negative form of the immune checkpoint    molecule.-   113. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the modified T cell comprises a first    targeting vector and a second targeting vector, the first targeting    vector comprising a nucleic acid sequence encoding a CAR binding    CD19 and the therapeutic agent, and the second targeting vector    comprises a nucleic acid sequence encoding a CAR biding UPK2, ACPP,    SIGLEC-15 or KISS1R and a dominant negative form of CD44.-   114. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the modified T cell comprises a first    targeting vector and a second targeting vector, the first targeting    vector comprising a nucleic acid sequence encoding a CAR binding a    blood antigen, and the second targeting vector comprises a nucleic    acid sequence encoding a CAR biding solid tumor antigen.-   115. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the modified T cell comprises a first    targeting vector and a second targeting vector, the first targeting    vector comprising a nucleic acid sequence encoding a CAR binding a B    cell antigen, and the second targeting vector comprises a nucleic    acid sequence encoding a CAR biding solid tumor antigen.-   116. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the solid tumor antigen is at least    one of antigens listed in Table 1, and/or the B cell antigen is    CD19, CD20, CD22, or BCMA.-   117. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the solid tumor antigen comprises at    least one of antigens listed in Table 1.-   118 A method of enhancing T cell expansion in a subject in need    thereof, the method comprising administering an effective amount of    the composition of T cells of any suitable preceding embodiments to    the subject, the subject having a higher level of T cell expansion    as compared with a subject that is administered an effective amount    of the CAR T cells that do not have the CAR binding the B cell    antigen.-   119. The nucleic acid, modified T cell or the method any suitable    preceding embodiments, wherein the modified T cell comprises a    nucleic acid sequence encoding hTERT, SV40LT, or a combination    thereof.-   120. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the modified T cell is more    proliferable than T cells without nucleic acid sequence.-   121. The isolated nucleic acid sequence, modified T cell or the    method of any suitable preceding embodiments, wherein the    proliferable cell remains functions of normal T cells/CAR T cells    such as cell therapy functions.-   122. The nucleic acid, modified T cell or the method any suitable    preceding embodiments, wherein the T cell comprises a CAR and is    cultured in the presence of an agent that is recognized by the    extracellular domain of the CAR, thereby producing a modified CAR    cell.-   123. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the integration of the nucleic acid    sequence encoding hTERT, the nucleic acid encoding SV40LT, or a    combination thereof includes genomic integration of the nucleic acid    sequence encoding hTERT, a nucleic acid encoding SV40LT, or a    combination thereof and constitutive expression of hTERT, SV40LT, or    a combination thereof.-   124. The nucleic acid sequence, modified T cell or the method of any    suitable preceding embodiments, wherein expression of hTERT, SV40LT,    or a combination thereof, is regulated by an inducible expression    system such as a rtTA-TRE system.-   125. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein modified T cell comprises a nucleic    acid sequence encoding a suicide gene such as a an HSV-TK system.-   126. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the cell has a reduced    graft-versus-host disease (GVHD) response in a bioincompatible human    recipient as compared to the GVHD response of the primary human T    cell.-   127. The nucleic acid, modified T cell or the method of any suitable    preceding embodiments, wherein the cell has reduced expression of    endogenous TRAC gene.-   128. A fusion protein comprising an extracellular domain, a    transmembrane domain, and an intracellular domain, the extracellular    domain derived from a first molecule, the intracellular domain    derived from a second molecule, the first molecule different from    the second molecule.-   129. A fusion protein comprising an extracellular domain, a    transmembrane domain, and an intracellular domain, the extracellular    domain derived from a first molecule, the intracellular domain    derived from a second molecule, the first molecule different from    the second molecule, wherein the first molecule comprises a    co-stimulatory domain, and the second molecule comprises a cytokine    receptor.-   130. An isolated nucleic acid encoding the fusion protein of    embodiments 128 or 129.-   131. A modified cell comprising the isolated nucleic acid of    embodiment 130 or the fusion protein of embodiments 128 or 129.-   132. A pharmaceutical composition comprising a population of the    modified cells of embodiment 131.-   133. A method of causing or promoting a T cell response in a subject    in need thereof and/or treating a tumor of the subject, the method    comprising administering an effective amount of the composition of    embodiment 132 to the subject.-   134. The method of embodiment 133, further comprising: administering    an effective amount of an agonist antibody binding the extracellular    domain.-   135. The method of embodiment 134, wherein the binding of the    extracellular domain with the agnostic antibody causes recruitment    of multiple first molecules into a receptor oligomer in which    signaling activation of the second molecule is triggered or    enhanced.-   136. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein the extracellular domain binds to an agonist antibody, and    wherein the agonist antibody is CDX 1140, SEA CD40, RO7009789, JNJ    64457107 (ADC1013), APX 005M, Chi Lob 7/4, TRX 518, MK 4166, MK    1248, GWN 323, INCAGN01876, BMS 986156, AMG 228, Tavolimab    (MED10562), PF 04518600, BMS 986178, MOXR 0916, GSK 3174998,    INCAGN01949, Utomilumab (PF 05082566), Urelumab (BMS 663513), GSK    3359609, JTX 2011, Theralizumab (TAB 08).-   137. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein the extracellular domain is an extracellular domain of a    co-stimulatory molecule.-   138. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein the extracellular domain is an extracellular domain of OX40,    CD40, 4-1BB, GITR, ICOS, CD28, CD27, HER2, or EGFR.-   139. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein: the extracellular domain is an extracellular domain of    CD40, GITR, OX40, 41BB, ICOS, or CD28;-   the co-stimulatory molecule is costimulatory molecule selected from    the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,    ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,    LIGHT, NKG2C, B7-H3, and any combination thereof; and/or the    intracellular is an intracellular domain of a cytokine receptor.-   140. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein the second molecule is OX40, CD40, 4-1BB, GITR, ICOS, CD28,    CD27, HER2, EGFR, IL10R, IL12R, IL18R1, IL23R, GP130, or IL15Ra.-   141. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-135,    wherein the intracellular domain is an intracellular domain of    IL12R, IL18R1, IL23R, GP130, or IL15Ra.-   142. The fusion protein, isolated nucleic acid, modified cell,    pharmaceutical composition, or method of one of embodiments 128-141,    wherein the transmembrane domain is an intracellular domain of OX40,    CD40, 4-1BB, GITR, ICOS, CD28, CD27, HER2, EGFR, IL12R, IL18R1,    IL23R, GP130, or IL15Ra.-   143. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-135, wherein the modified cell is lymphocyte,    leukocyte, or PBMC; or cells, NK cells, T cells, or dendritic cells.-   144. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-143, wherein the modified cell further comprises    a Chimeric antigen receptor (CAR) or a modified TCR.-   145. The modified cell, pharmaceutical composition, or method of    embodiment 144, wherein the TCR is a modified TCR.-   146. The modified cell, pharmaceutical composition, or method of    embodiment 144, wherein the TCR is derived from spontaneously    occurring tumor-specific T cells in patients.-   147. The modified cell, pharmaceutical composition, or method of    embodiment 144, wherein the TCR binds a tumor antigen.-   148. The modified cell, pharmaceutical composition, or method of    embodiment 147, wherein the tumor antigen comprises CEA, gp100,    MART-1, p53, MAGE-A3, or NY-ESO-1, or the TCR comprises TCRγ and    TCRδ Chains or TCRα and TCRβ chains, or a combination thereof.-   149. The modified cell, pharmaceutical composition, or method of    embodiment 144, wherein the CAR comprises an extracellular domain, a    transmembrane domain, and an intracellular domain, and wherein the    extracellular domain binds an antigen.-   150. The modified cell, pharmaceutical composition, or method of    embodiment 149, wherein the intracellular domain comprises a    costimulatory signaling region that comprises an intracellular    domain of a costimulatory molecule selected from the group    consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,    lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,    NKG2C, B7-H3, and any combination thereof.-   151. The modified cell, pharmaceutical composition, or method of    embodiment 150, wherein the antigen is Epidermal growth factor    receptor (EGFR), Variant III of the epidermal growth factor receptor    (EGFRvIII), Human epidermal growth factor receptor 2 (HER2),    Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA),    Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),    Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase    IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125    (CA125), Cluster of differentiation 133 (CD133), Fibroblast    activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1    (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,    GUCY2C, CD207, CD3, CDS, B-Cell Maturation Antigen (BCMA), or CD4.-   152. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-151, wherein the modified cell or the T cells    comprise an additional CAR binding a solid tumor antigen, and the    CAR binds an antigen of a white blood cell.-   153. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-151, wherein the modified cell or the T cells    comprise a dominant negative PD-1.-   154. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-151, wherein the modified cell or the T cells    comprise a modified PD-1 lacking a functional PD-1 intracellular    domain.-   155. The modified cell, pharmaceutical composition or method of one    of embodiments 131-154, wherein the modified cell further comprises    a nucleic acid encoding therapeutic agent.-   156. The modified cell, pharmaceutical composition, or method of    embodiment 155, wherein the isolated nucleic acid comprises a    promoter comprising a binding site for a transcription modulator    that modulates the expression and/or secretion of the therapeutic    agent in the cell.-   157. The modified cell, pharmaceutical composition, or method of    embodiment 156, wherein the transcription modulator is or includes    Hif1a, NFAT, FOXP3, and/or NFkB.-   158. The modified cell, pharmaceutical composition, or method of    embodiment 157, wherein the promoter is responsive to the    transcription modulator.-   159. The modified cell, pharmaceutical composition, or method of    embodiment 157, wherein the promoter is operably linked to the    nucleic acid encoding the therapeutic agent such that the promoter    drives expression and/or secretion of the therapeutic agent in the    cell.-   160. The modified cell, pharmaceutical composition, or method of    embodiment 157, wherein expression of the therapeutic agent is    regulated by an inducible gene expression system.-   161. The modified cell, pharmaceutical composition, or method of    embodiment 160, wherein the inducible gene expression system    comprises or is a lac system, a tetracycline system, or a galactose    system.-   162. The modified cell, pharmaceutical composition, or method of    embodiment 160, wherein the inducible gene expression system    comprises or is a tetracycline system.-   163. The modified cell, pharmaceutical composition, or method of    embodiment 162, wherein the inducible gene expression system    comprises or is a tetracycline on system, and an inducer is    tetracycline, doxycycline, or an analog thereof.-   164. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-163, wherein the modified cell is a T cell    derived from a primary human T cell isolated from a human donor.-   165. The modified cell, pharmaceutical composition, or method of    embodiment 164, wherein the cell has a reduced expression of    endogenous TRAC gene.-   166. The modified cell, pharmaceutical composition, or method of one    of embodiments 131-163, wherein the modified cell is a T cell    derived from a primary human T cell isolated from a subject having    cancer.

The related sequences are provided in Table 2 as well as PCT PatentApplications Nos: PCT/CN2016/075061, PCT/CN2018/08891, andPCT/US19/13068, which are incorporated herein by reference in theirentirety.

EXAMPLES

Lentiviral vectors that encode individual CAR molecules were generatedand transfected into T cells, which are elaborated below. Techniquesrelated to cell cultures, construction of cytotoxic T lymphocyte assaycan be found in “Control of large, established tumor xenografts withgenetically retargeted human T cells containing CD28 and CD137 domains,”PNAS, Mar. 3, 2009, vol. 106, no. 9, 3360-3365 and “Chimeric ReceptorsContaining CD137 Signal Transduction Domains Mediate Enhanced Survivalof T Cells and Increased Antileukemic Efficacy In Vivo,” MolecularTherapy, August 2009, vol. 17, no. 8, 1453-1464, which are incorporatedherein by reference in their entirety. FIG. 6 shows flow cytometryresults of T cells comprising a polynucleotide encoding CD19CAR (scFvbinding CD19: SEQ ID NO: 5) and CD19CAR-2a-DNM1L (SEQ ID NO: 118) thathave been cultured to Day 5. Sequence information corresponding tovarious vectors in the Examples are provided in Table 2.

FIGS. 7-10 show CAR expression and functions of T cells comprising a CARand T cells comprising a CAR and secretable SIGLEC-15 scFv. T cells fromvarious donors were obtained and transfected with vectors comprisingCD19 CAR (SEQ ID NO: 5) or CD19 CAR-anti-SIGLEC-15 scFvs (SEQ ID NO: 115or 116). As shown in FIG. 7, transduction of polynucleotides encodinganti-SIGLEC-15 scFvs did not affect the expression of CAR, whichindicates that CAR expression is normal. Further, as shown in FIG. 8, Tcells comprising polynucleotides encoding anti-SIGLEC-15 SCFV-1 andSCFV-2 have significant mRNA expression compared to control.

FIG. 9 shows flow cytometry results of the gene expression of tumorcells. The tumor cells comprising SIGLEC-15 and CD19+ does not expressSIGLEC-15, while the tumor cells comprising CD19+ and SIGLEC-15+ expressSIGLEC-15. FIG. 10 shows the killing function assay of T cellscomprising CD19CAR and T cells comprising CD19CAR and secretableSIGLEC-15 scFv. Under different E:T ratios, various CART cells wereco-cultured with SIGLEC-15-CD19+ and SIGLEC-15+-CD19+ tumor cells, andthe function was determined by flow cytometry. As shown in FIG. 10, Tcells comprising CD19 CAR and secretable SIGLEC-15 scFv showed enhancedkilling of CD19+ and SIGLEC15+ tumor cells than T cells comprising CD19CAR alone. These results demonstrate that secretable anti-SIGLEC-15 scFvblocked the SIGLEC-15-CD44 pathway to enhance the T cells' function.

All publications, patents and patent applications cited in thisspecification are incorporated herein by reference in their entiretiesas if each individual publication, patent or patent application werespecifically and individually indicated to be incorporated by reference.While the foregoing has been described in terms of various embodiments,the skilled artisan will appreciate that various modifications,substitutions, omissions, and changes may be made without departing fromthe spirit thereof.

1. A modified cell comprising a polynucleotide encoding an antibodybinding SIGLEC-15, wherein the antibody is secretable or attached to themembrane of the modified cell.
 2. The modified cell of claim 1, whereinthe polynucleotide comprises a polynucleotide encoding at least one ofSEQ ID NOS: 115, 116, 117, 118, or
 119. 3. The modified cell of claim 1,wherein the antibody is a secretable scFv.
 4. The modified cell of claim3, wherein the polynucleotide comprises a polynucleotide encoding SEQ IDNO: 115 or
 116. 5. The modified cell of claim 3, wherein the modifiedcell further comprises a dominant negative form of an immune checkpointinhibitor selected from the group consisting of programmed death 1(PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyteattenuator (BTLA), T cell immunoglobulin mucin-3 (TIM-3),lymphocyte-activation protein 3 (LAG-3), T cell immunoreceptor with Igand ITIM domains (TIGIT), leukocyte-associated immunoglobulin-likereceptor 1 (LAIRI), CD44, natural killer cell receptor 2B4 (2B4), and CD160.
 6. The modified cell of claim 3, wherein the modified T cell isengineered to express and secrete a therapeutic agent such as acytokine.
 7. The modified cell of claim 6, wherein the therapeutic agentcomprises at least one of IL-33, IL-1β, TNFα, MALP-2, IL1, IL17, G-CSFor GM-CSF, IL-6, IL-12, and IFN-γ.
 8. The modified cell of claim 6,wherein the therapeutic agent is regulated by Hif1a, NFAT, FOXP3, and/orNFkB.
 9. The modified cell of claim 3, wherein the modified cellcomprises an antigen-binding molecule binding a tumor antigen, and themodified cell is a T cell derived from a healthy donor or a subjecthaving cancer.
 10. The modified cell of claim 9, wherein theantigen-binding molecule is a CAR, which comprises an antigen-bindingdomain, a transmembrane domain, and an intracellular signaling domain.11. The modified cell of claim 10, wherein the antigen-binding domainbinds a tumor antigen selected from a group consisting of: TSHR, CD19,CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA,Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3,KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24,PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1,EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGSS, HMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRCSD,CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1,LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, surviving, telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, andIGLL1.
 12. The modified cell claim 11, wherein the intracellularsignaling domain comprises a co-stimulatory signaling domain, or aprimary signaling domain and a co-stimulatory signaling domain, whereinthe co-stimulatory signaling domain comprises a functional signalingdomain of a protein selected from the group consisting of CD27, CD28,4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta,IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a,LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1,ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44,NKp30, NKp46, and NKG2D
 13. The modified cell of claim 9, wherein theantigen-binding molecule is a modified TCR.
 14. The modified cell ofclaim 13, wherein the TCR is derived from spontaneously occurringtumor-specific T cells in patients.
 15. The modified cell of claim 14,wherein the TCR binds to a tumor antigen.
 16. The modified cell of claim15, wherein the tumor antigen comprises CEA, gp100, MART-1, p53,MAGE-A3, or NY-ESO-1.
 17. The modified cell of claim 15, wherein the TCRcomprises TCRγ and TCRδ chains, TCRα and TCRβ chains, or a combinationthereof.
 18. The modified cell of claim 3, wherein the cell is an immunecell.
 19. The modified cell of claim 18, wherein the immune cell is a Tcell or an NK cell.
 20. The modified cell of claim 19, wherein theimmune effector cell is a CD4+ T cell, a CD8+ T cell, or a combinationthereof.